Biol Trace Elem Res DOI 10.1007/s12011-010-8701-6
Investigating the Effect of Heavy Metals on Developmental Stages of Anther and Pollen in Chenopodium botrys L. (Chenopodiaceae) Nafiseh Yousefi & Abdolkarim Chehregani & Behrouz Malayeri & Bahareh Lorestani & Mehrdad Cheraghi
Received: 18 February 2010 / Accepted: 8 April 2010 # Springer Science+Business Media, LLC 2010
Abstract Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. Preliminary studies have shown that Chenopodium botrys can grow in some heavy metal contaminated soils and is a high accumulator plant species for Cu and moderately accumulator plant species for Fe, Mn, and Zn, thus, was considered as an important species in this study. Based on that, in this species, we studied the individual effects of heavy metals on the formation, development, and structure of anther and pollen. To achieve this purpose, surrounding area of Hame-Kasi iron and copper mine (Hamedan, Iran) was chosen as a polluted area where the amount of some heavy metals was several times higher than the natural soils. Flowers and young pods were removed from nonpolluted and polluted plants, fixed in FAA 70, and subjected to developmental studies. Analysis of anther development in plants from contaminated sites showed general similarities in the pattern of pollen formation with those from non-polluted ones, but also deviation from typical form of major stages of anther and pollen development was seen in plants from polluted ones. Stabilizing of tapetum layer, increasing in tapetum layer numbers, thickening callose wall in the microspore mother cell stage, changing the anther shape, and decreasing the size of anther were the effects of heavy metals. Reduction of pollen number was also seen in the plants collected from polluted area. Keywords Anther development . Chenopodium botrys L. . Heavy metals . Pollen grain . Pollution
N. Yousefi (*) : B. Lorestani : M. Cheraghi Islamic Azad University-Hamedan Branch, Hamedan, Iran e-mail:
[email protected] A. Chehregani : B. Malayeri Department of Biology, Bu-Ali Sina University, Hamedan, Iran
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Introduction Heavy metals make a significant contribution to environment as a result of human activity such as mining, smelting, electroplating, energy fuel producing, power transmitting, intensive agriculturing, sludge dumping, and melting operations [1]. In trace amounts, several of these ions are required for metabolism, growth, and development. However, problems arise when cells are confronted with an excess of these vital ions or with nonnutritional ions that lead to cellular damage [2]. Although plants can adapt rather readily to chemical stress, they also may be very sensitive to an excess of particular trace element. Visible symptoms of toxicity vary for each plant species and even for individual plants, but most common and non-specific symptoms are inactivation of biomolecules by either blocking essential functional groups or by displacing essential metal ions [3], changes in permeability of the cell membrane, reactions of thiol groups with cations and damage to photosynthesis apparatus [4]. Researchers have observed that some plant species are endemic to metalliferous soils and can tolerate greater than usual amounts of heavy metals or other toxic compounds [5– 8]. Although several studies have been conducted in order to evaluate the effects of different heavy metal concentrations on live plants [8–10], there are few reports about toxic effects of heavy metals on the developmental stages of sex organs [11, 12]. The importance to study the interaction between pollens and environmental pollution has been put forward two decades ago by Ishizaki et al. [13] who demonstrated the relationship between pollutants and a higher prevalence of pollinosis in urban areas. Since then, effects of pollution on the molecular and developmental biology of pollen have been the subject of interesting studies revealing the accumulation of numerous inorganic elements such as sulfur, cadmium, and lead in pollen grains [14–17] and the acidification of pollen surfaces by the absorption of acid gasses such as nitric acid in polluted areas [18]. Behrendt et al. [14], Sawidis [17] and Sawidis, and Reiss [19] reported that pollen grains can accumulate heavy metals, i.e. lead and cadmium, but also nitrate and other compounds. Histological examination of plant tissues has been a useful tool for diagnosing the sensitivity of plants to pollutants [20]. The present manuscript reports data regarding effect of heavy metal stress on development of anther and pollen in Chenopodium botrys in media containing Fe, Cu, Mn, and Zn ions.
Materials and Methods Sampling Area Characterization In this study, a known metal-contaminated site located in a square (N 34° 57′ 16″ and E 48° 8′ 26″), (N 34° 56′ 14″ and E 48° 8′ 22″), (N 34° 55′ 58″ and E 48° 11′ 34″) and (N 34° 54′ 53″ and E 48° 10′ 25″) in the surrounding area of Hame-Kasi mine of northwest Hamadan province in Iran, was chosen as a polluted area (Fig. 1). The site has occupied approximately 10,000 m2 and is covered mainly by grasses. Human activities such as mining have contributed to elevated metal concentrations in this site. In this region, the amount of some heavy metals was several times higher than those in out of mine. As shown in Table 1, the amounts of Fe, Mn, Zn, and Cu in soil of polluted region were, respectively, 16.3, 12, 12.5, and 11.1 times higher than those in non-polluted region.
Effect of Heavy Metals on Anther and Pollen in Chenopodium botrys L.
Fig. 1 The position of Hame-Kasi mine in Iran country
Sampling Species Characterization In this study, C. botrys grown in the vicinity of the Hame-Kasi iron and copper mine was chosen to study the effect of heavy metal stress on pollen and anther development because Malayeri et al. [21] reported that C. botrys is a high accumulator plant for Cu and a moderately accumulator plant for Fe, Mn, and Zn and can absorb a wide range of soil metals (Cu, Fe, Mn, and Zn). Also according to findings of Nouri et al. [22] C. botrys accumulates 185 and 150 μg/g Cu and 177.3 and 1,288 μg/g Mn in its root and shoot, respectively, which is higher than other species grown in the surrounding area of HameKasi mine (Fig. 2).
Table 1 Comparison of Concentrations of Metals (micrograms per gram) in Soils Collected from Mining Site as a Polluted Area and Out of the Mine as a Non-Polluted Area Heavy metal Fe
Non-polluted site 2,078±25.1
Polluted site 33,890±1,565.1
Mn
32±9.8
385.3±23.8
Zn
112±30.7
1,405±137
Cu
10.7±1.5
119±31.5
Data presented are the means±SE of five sampling sites in polluted and non-polluted areas. The total metal concentrations in polluted soils are very high, and the amount of Fe, Mn, Zn, and Cu in polluted areas is several times higher than non-polluted areas. Adapted from Malayeri et al. [21]
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Fig. 2 Comparison of concentrations of metals (micrograms per gram) in C. botrys collected from mining site as a polluted area and out of the mine as a non-polluted area. Data presented are the means±SE of five specimens in polluted and non-polluted areas. The amount of Fe, Mn, Zn, and Cu in polluted area is several times higher than that of non-polluted area. Adapted from Malayeri et al. [21] and Nouri et al. [22]
C. botrys was collected from January to June in 2007. A voucher specimen was placed in Bu-Ali Sina Herbarium (BASUH, 1037), Botany Department at the same university and labeled as follows: Iran, province of Hamedan, 35 km from Hamedan to Kordestan, alt. 2,386 m. Developmental Studies For the characterization of heavy metal effects on developmental stages of anther, flowers, and young buds were removed from the naturally growing plants in the polluted and nonpolluted areas, separately. The specimens were fixed in FAA (formaldehyde, glacial acetic acid, and 70% ethanol, 5:5:90), stored in 70% ethanol, embedded in paraffin, and sectioned at 8 µm with a Leitz 1512 microtome. Staining was carried out with Mayer haematoxilin– eosin method [23]. At each developmental stage, at least 20 flowers were studied under a light microscope Zeiss Axiostar Plus and differentiations between polluted and non-polluted plants were analyzed.
Results and Discussion Heavy metals are potentially highly toxic to all organisms including animals and plants [11]. These elements have recently received the attention of researchers all over the world, mainly due to their harmful effects on plant. The toxic effects of metals have also been intensively studied at the level of biochemical–physiological–histological process such as photosynthesis [24], transpiration [25], enzyme activity [26], metal accumulation in tissue [27] or developmental process of sex organs [11, 12]. A survey of microscopic specimens prepared from plants collected from polluted area, showed that major stages of anther and pollen development in these plants was the same as those from non-polluted ones, but there were some differences between polluted and nonpolluted plants. In this paper, we avoided to describe anther and pollen developmental stages in non-polluted plants and focused on description of abnormalities that were seen in
Effect of Heavy Metals on Anther and Pollen in Chenopodium botrys L.
plants collected from polluted area. Our data showed that the anther in C. botrys is dithecous and tetralocular and its development being of the dicotyledonous type that is in accordance with some prior findings [28, 29]. As shown in Fig. 3, the anther wall comprises epidermis, one or two middle layer and a single-layered tapetum. Also, it has a column of sterile connective tissue. The pollen grains in C. botrys are radial symmetrical, polyporate, and spherical, which is in agreement with the results by Pinar and Inceoglu [30]. The wall of the mature pollen grain comprises two principal intine and exine layers (Fig. 4). According to our results, in plants grown in the mine area, some abnormalities were seen during anther development. Slow degradation of tapetum layer was observed in plants collected from polluted area than in non-polluted ones, so that this layer was retained to final stages of anther development (comparison of Fig. 5a, b). Probably, the reason of this effect is more stability of tapetum layer that can cause to inhibition of heavy metal penetration into the pollen grains. Typically, the tapetum is a single layer of cells [28], but, significantly, increase in tapetum layer numbers was observed in plant collected from polluted area, so that it is composed of about four layers (comparison of Fig. 6a, b). Since tapetum is of considerable physiological importance [28], thus, with increasing in the wall layer of cells, accumulation, and inactivation of heavy metals occurred that are probably related to their binding in cell walls, compartmentalization in vacuoles, and complexation with metal binding proteins and peptides, especially phytochelatin and metallothioneins [31]. In the microspore mother cell stage, a thin layer of callose is deposited beneath the microspore mother cell coat (Fig. 7a), but, as shown in the Fig. 7b, microspore mother cells are characterized by a thickened callose wall in polluted plants. Callose wall can considered as a final barrier against the penetration of heavy metals toward the pollen. In addition, the shape of polluted plant anther was changed dramatically in polluted plants (comparison of Fig. 8a, b), consistent with the observations of Malayeri et al. [11] and Majd and Chehregani [32] about the effect of heavy metals and other environmental toxins and pollutants on number of ovules. It was observed that the pollen grain number was significantly decreased in plant collected from polluted area (comparison of Fig. 8a, b), consistent with the observations of
Fig. 3 Transverse section of a tetrasporangiate anther with various tissues in C. botrys, ta tapetum, me middle layer, ep epidermis, co connective. Bar=150 μm
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Fig. 4 Morphology of mature releasable pollen grains in C. botrys, ex exine, in intine. Bar=400 μm
Malayeri et al. [11] and Majd and Chehregani [32] about effect of heavy metals and other environmental toxins and pollutants on ovule number. As the last effect of heavy metals, in some samples, anther could not complete its growth and became smaller in plants collected from polluted area than in non-polluted ones, with a maximum decrease of 30% (data not shown). Probably, the reason of this effect is more the stability of the wall layers that does not permit the growth of anther. Similar results have been reported by Yousefi et al. [12] about the effect of heavy metals, and Chehregani and Kavianpour [33] and Chehregani et al. [34] about the effect of other environmental pollutants such as acid rain and diesel exhaust particles on the ovules and embryo sac development. Apparently, these processes are strategies employed by plants, at least in part, to face unavoidable stress conditions. Heavy metals, as an important environmental pollutant, have different morphological, biochemical, and cytological effects on plants [11, 35–38]. Based on the results, we concluded that the anther development of C. botrys is seriously affected by heavy metals. The results of this study indicated that heavy metals can induce several abnormalities on the anther development, thus affecting the fertility and survival of plants. Fig. 5 Transverse section through distinct anther, collected from polluted and non-polluted areas. a Column b indicates the normal anther from non-polluted region and b column indicates the abnormalities in anther from polluted plants. Blockage of the tapetum layer degradation (arrow) in plant collected from polluted area (compared a with b). Bar=250 μm in a, b Fig. 6 Transverse section through distinct anther, collected from polluted and non-polluted areas. (a) Column indicates the normal anther from non-polluted region and (b) column indicates the abnormalities in anther from polluted plants. Significant increase in tapetum layer numbers (arrow) in plant collected from polluted area (compared a with b). Bar=250 μm in a and b Fig. 7 Transverse section through distinct anther, collected from polluted and non-polluted areas. a Column indicates the normal anther from non-polluted region and b column indicates the abnormalities in anther from polluted plants. Faster growth of callose layer (arrow) in plant collected from polluted area (compared a with b). Bar=300 μm in a; 400 μm in b Fig. 8 Transverse section through distinct anther, collected from polluted and non-polluted areas. a Column indicates the normal anther from non-polluted region and b column indicates the abnormalities in anther from polluted plants. Changing the anther shape and reduction of pollen number in plant collected from polluted area (compared a with b). Bar=100 μm a and 150 μm in b
Effect of Heavy Metals on Anther and Pollen in Chenopodium botrys L.
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This report is the first to provide detailed effects of heavy metals on the developmental stages of anther and pollen. Acknowledgement The authors would like to express their appreciation to Bu-Ali Sina University and Islamic Azad University (Hamedan, Iran) for their facilities and kind support.
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